During the past year we have made significant progress in four major areas of investigation using our Novikoff hepatoma system. The first involves further evaluation of the relationship between junctional permeability to Lucifer yellow, as determined with computer-assisted video analysis, and cell size. We have found that permeability increases roughly in proportion to cell volume over the range appropriate for most cells. Thus it may be possible that a cytoplasmic factor, produced in proportion to cell volume, may regulate junctional area, or that junctions between younger cells may have a lower conductance/channel than those between older cells. The second involves testing longer term effect of 4~ incubation on junctional formation. In contrast with shorter times (15 min), longer times at low temperature result in a substantial reduction in junctional areas and apparently junctional maturation. This result is consistent with a temperature-dependent reduction in migration of intramembranous junctional precursors over long distances and fits with our hypothetical scheme for junctional maturation. The third involves completion of our analyses of filipin binding to newly formed junctions. It is now clear that formation plaques, with or without junctional aggregates, fail to show evidence of filipin-cholesterol deformations. This may mean that the plaques are deficient in cholesterol or that they are more rigid than non-junctional membrane. The latter possibility would be consistent with the small effect of low temperature on the initiation of junction formation. The fourth area involves attempts at isolating gap junctions from Novikoff cells. A new procedure developed in J.P. Revel's lab has given some success in isolating gap junctions, although the SDS gels still are quite complex. Further work along these lines is planned. (A)